Atrial Fibrillation: unravelling the significance of lysosomes in atria under normal and pathological conditions

Lysosomes are responsible for the degradation of intracellular and extracellular macromolecules. They also play roles in signalling relevant to cardiac function and cardiac disease, however, the extent of altered lysosomal function in Atrial fibrillation (AF) is unknown. This thesis describes the development of a fractionation method and label-free quantitative proteomics using guinea-pig atrial tissue lysate (TL), endo-lysosome (EL) and mitochondria (Mito) fractions. Over two thousand proteins were detected, of which 564 were more abundant in EL than TL. The resulting lysates contained atrial, mitochondrial and EL-specific proteins. Significant EL proteins recovered included catalytic activity proteins. Protein enrichment and networks were studied. The developed fractionation method was applied to goat left atrial AF and sham control samples, including network and pathway analysis. Regulated proteins discovered by proteomics were validated with Western blotting, including Ras-related protein 11 (RAB11). In addition, lysosomal assays (beta-galactosidase and beta-hexosaminidase) were performed. Out of 2104 proteins, levels of 340 proteins in TL, 113 in mito and 148 in EL were significantly altered. EL fractions showed enrichment for EL specific proteins including GAA, clathrin light chain B (CLTB), T-complex protein 1 subunit beta (CCT2), and vacuolar protein-sorting-associated protein 25 (VPS25). KEGG pathways analysis highlighted mitochondrial oxidative-phosphorylation OXPHOS, and AMPK pathway protein upregulation, indicating a potential increased ATP energy demand in AF samples. Upregulation of protein processing suggests increased vesicular trafficking, potentially related to increased metabolic energy demand. The downregulation of autophago-lysosome fusion proteins such as dynein, dynactin and dynamin indicate a possible disruption in the cellular degradation of macromolecules during chronic AF. The data presented in this thesis therefore provides evidence to support the role of the lysosome as an important organelle that may play a role in AF pathology